Understanding molecular mechanisms driving a pluripotent progenitor cell to differentiate into a specific cell type is germane to the study of organogenesis and has important significance in regenerative medicine. While the roles of key cardiac-specific transcription factors in heart development have been carefully studied, the importance for protein complexes involved in epigenetic regulation or transcription elongation in cardiac cell formation is just being revealed. In this proposal, we aim to investigate molecular mechanisms by which the RNA Polymerase II- Associated Factor 1 Complex (PAF1C) controls heart development. Biochemical and genetic studies in multiple model organisms suggest that PAF1C functions as a transcription platform required for critical signaling events. However, its roles in heart formation are not known. Our recent genetic studies discovered a novel and essential role for Rtf1, a component of the PAF1C, in the formation and proliferation of cardiomyocytes. We found that zebrafish rtf1 deficient embryos lack the entire population of cardiac progenitor cells, demonstrating that Rtf1 is absolutely required for heart development. Our preliminary data obtained from structure-function analysis and loss- and gain-of- function studies lead us to hypothesize that Rtf1 controls cardiac gene expression by PAF1C- associated epigenetic modification and PAF1C-independent transcription regulation. We will examine this hypothesis in zebrafish and evaluate how these mechanisms influence the formation of cardiac progenitor cells and the proliferation of cardiomyocytes (Aim 1). We showed that Rtf1 promotes cardiac differentiation from embryonic mesoderm in zebrafish. We will examine whether this mechanism is conserved in mammals using mouse ES cells as an in vitro differentiation model. We will also create cardiac-specific conditional knockout mice to assess the role of Rtf1 in mouse heart development (Aim 2). Finally, many developmentally regulated genes are reutilized during heart regeneration and Tbx20, an Rtf1 downstream transcription factor, is upregulated after ventricular resection. We thus propose to examine whether the Rtf1-Tbx20 pathway is involved in heart regeneration using both adult zebrafish and neonatal mouse heart regeneration models (Aim 3). Successful completion of the proposed projects will provide new mechanistic insights into the regulation of cardiac progenitor cell formation and cardiomyocyte proliferation during development and in regeneration.